Modulation of P2X4 pore closure by magnesium,potassium, and ATP

Abstract

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+.